WO2016092045A1

WO2016092045A1 - Methods and kits for predicting medically refractory acute severe colitis

Info

Publication number: WO2016092045A1
Application number: PCT/EP2015/079315
Authority: WO
Inventors: Eric Ogier-Denis; Yoram BOUHNIK; Xavier Treton; Gilles WAINRIB; Mathieu UZZAN; David LAHARIE; Ian MORILLA
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université Paris Diderot - Paris 7; Université Paris Xiii Paris-Nord; Assistance Publique-Hôpitaux De Paris (Aphp); Université De Bordeaux; Chu De Bordeaux
Priority date: 2014-12-11
Filing date: 2015-12-10
Publication date: 2016-06-16

Abstract

The present invention relates to methods and kits for predicting medically refractory acute severe colitis. In particular, the present invention relates to a method for predicting medically refractory acute severe colitis in a subject comprising the steps consisting of i) determining in a sample obtained from the subject the expression levels of miR-1825, miR- 938, miR-4699, miR-23c, miR-4659a-3p, miR-4477a, miR-320b-1, miR-3128, and miR- 4755, comparing the expression levels determined at step i) with their corresponding predetermined reference values and ii) concluding that the subject has a high risk of having medically refractory acute severe colitis when the expression level of miR-1825 and miR-938 are lower than their respective predetermined reference values and when the expression levels of miR-4699, miR-23c, miR-4659a-3p, miR-4477a, miR-320b-1, miR-3128, and miR- 4755 are higher than their respective predetermined reference values, or concluding that the subject has a low risk of having medically refractory acute severe colitis when the expression level of miR-1825 and miR-938 are higher than their respective predetermined reference values and when the expression levels of miR-4699, miR-23c, miR-4659a-3p, miR-4477a, miR-320b-1, miR-3128, and miR-4755 are lower than their respective predetermined reference values.

Description

METHODS AND KITS FOR PREDICTING MEDICALLY REFRACTORY ACUTE

SEVERE COLITIS

FIELD OF THE INVENTION:

The present invention relates to methods and kits for predicting medically refractory acute severe colitis.

BACKGROUND OF THE INVENTION:

Ulcerative colitis is an inflammatory disease of the colon where the erosion or ulcer is formed in the mucous membrane of the large intestine. The characteristic symptoms may be diarrhea with or without melena and frequent abdominal pain. The lesion has the property of extending continuously to the ascending (orifice side) from the rectum, further to the entire colon from the rectum in maximum. One of the serious complications occurring in 15-25% of subjects is severe acute colitis. Acute severe colitis (ASC) is potentially life-threatening. The diagnosis of ASC is defined according to Truelove's original criteria as a bloody stool frequency > 6 per day and at least one of the following: pulse > 90 beats per minute, temperature > 37.8 °C, haemoglobin < 10.5 g/dL, or an ESR > 30 mm/h. Effective clinical management of active ACS requires a comprehensive understanding of the disease extent, the severity and the potential risks and benefits of the available interventions. Options for the treatment of acute severe ulcerative colitis have broadened with the use of ciclosporin and infliximab, but corticosteroids remain the cornerstone of initial therapy. However a third of subjects will fail to respond, and further management involves critical and timely decisions on whether to use rescue therapy in the form of immunomodulatory drugs such as ciclosporinciclosporin A or anti-TNF therapies such as Infliximab. When it is considered that the subject is medically refractory, colectomy is unavoidable. Accordingly, determining criteria on admission associated with a poor outcome (i.e. medically refractory) are thus needed to help clinicians for advising subjects when managing acute severe colitis. SUMMARY OF THE INVENTION:

The present invention relates to methods and kits for predicting medically refractory acute severe colitis. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION: Acute severe ulcerative colitis remains a significant clinical challenge and the ability to predict, at an early stage, those individuals at risk of colectomy for medically refractory would be a major clinical advance. As disclosed herein, the inventors identify a set of 9 miRNAs that are capable to discriminate subject who will medically refractory from those that will not be medically refractory.

Accordingly, the present invention relates to a method for predicting medically refractory acute severe colitis in a subject comprising the steps consisting of i) determining in a sample obtained from the subject the expression levels of miR-1825, miR-938, miR-4699, miR-23c, miR-4659a-3p, miR-4477a, miR-320b-l , miR-3128, and miR-4755, comparing the expression levels determined at step i) with their corresponding predetermined reference values and ii)

concluding that the subject has a high risk of having medically refractory acute severe colitis when the expression level of miR-1825 and miR-938 are lower than their respective predetermined reference values and when the expression levels of miR-4699, miR-23c, miR-4659a-3p, miR-4477a, miR-320b-l, miR-3128, and miR-4755 are higher than their respective predetermined reference values, or concluding that the subject has a low risk of having medically refractory acute severe colitis when the expression level of miR-1825 and miR-938 are higher than their respective predetermined reference values and when the expression levels of miR-4699, miR-23c, miR-4659a-3p, miR-4477a, miR-320b-l, miR-3128, and miR-4755 are lower than their respective predetermined reference values

As used herein, the expression "medically refractory acute severe colitis" is defined as acute severe colitis requiring colectomy for symptoms uncontrolled by immunosuppressive drugs. The expression "medically refractory acute severe colitis" also refers to medically non- responsive acute severe colitis requiring colectomy. The expression "medically refractory acute severe colitis" also refers to acute severe colitis that will not respond to immunosuppressive drug treatment and therefore requiring colectomy. The acute severe colitis activity can be measured according to the standards recognized in the art. The acute severe colitis activity may be measured by clinical and physical examination, Lichtiger score, and histological grading. As used herein, the term "immunosuppressive drug" refers to any substance capable of producing an immunosuppressive effect, e.g., the prevention or diminution of the immune response. The typical medically regimen of acute severe colitis include in first lines administration of corticosteroid and in second lines administration with anti-TNFa drug or immunosuppressive drugs such as ciclosporin.

As used, the term "corticosteroids" has its general meaning in the art and refers to class of active ingredients having a hydrogenated cyclopentoperhydrophenanthrene ring system endowed with an anti-inflammatory activity. Corticosteroid drugs typically include cortisone, Cortisol, hydrocortisone (1 ip,17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene, 3,20- dione disodium), dihydroxy cortisone, dexamethasone (21-(acetyloxy)-9-fluoro-ip,17- dihydroxy-16a-m-ethylpregna-l,4-diene-3,20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-l 1-β, 17,21, trihydroxy- 16P-methylpregna-l,4 diene-3,20-dione 17,21 -dipropionate). Other examples of corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort and betamethasone, corticosteroids, for example, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.

As used herein, the term "anti-TNFa drug" is intended to encompass agents including proteins, antibodies, antibody fragments, fusion proteins (e.g., Ig fusion proteins or Fc fusion proteins), multivalent binding proteins (e.g., DVD Ig), small molecule TNFa antagonists and similar naturally- or normaturally-occurring molecules, and/or recombinant and/or engineered forms thereof, that, directly or indirectly, inhibit TNFa activity, such as by inhibiting interaction of TNFa with a cell surface receptor for TNFa, inhibiting TNFa protein production, inhibiting TNFa gene expression, inhibiting TNFa secretion from cells, inhibiting TNFa receptor signaling or any other means resulting in decreased TNFa activity in a subject. The term "anti-TNFa drug" preferably includes agents which interfere with TNFa activity. Examples of anti-TNFa drugs include, without limitation, infliximab (REMICADE™, Johnson and Johnson), human anti-TNF monoclonal antibody adalimumab (D2E7/HUMIRA™, Abbott Laboratories), etanercept (ENBREL™, Amgen), certolizumab pegol (CIMZIA®, UCB, Inc.), golimumab (SIMPONI®; CNTO 148), CDP 571 (Celltech), CDP 870 (Celltech), as well as other compounds which inhibit TNFa activity, such that when administered to a subject in which TNFa activity is detrimental, the disorder (i.e. acute severe colitis) could be treated.

Immunosuppressive drugs also include, without limitation, ciclosporin, thiopurine drugs such as azathioprine (AZA) and metabolites thereof; anti-metabolites such as methotrexate (MTX); sirolimus (rapamycin); temsirolimus; everolimus; tacrolimus (FK-506); FK-778; anti-lymphocyte globulin antibodies, anti-thymocyte globulin antibodies, anti-CD 3 antibodies, anti-CD4 antibodies, and antibody-toxin conjugates; ; mycophenolate; mizoribine monophosphate; scoparone; glatiramer acetate; metabolites thereof; pharmaceutically acceptable salts thereof; derivatives thereof; prodrugs thereof; and combinations thereof. In particular, ciclosporin (also named "ciclosporin" A or "CyA" was the cornerstone of rescue medical therapy for steroid-refractory acute severe colitis until the advent of anti-TNF agents. CyA is a competitive calcinuerin inhibitor with potent immunosuppressive properties.

As used herein, the term "colectomy" refers to surgical resection of any extent of the large intestine (colon). Herein, colectomy includes, but is not limited to, right hemicolectomy, left hemicolectomy, extended hemicolectomy, transverse colectomy, sigmoidectomy, proctosigmoidectomy, Hartmann operation, "double-barrel" or Mikulicz colostomy, total colectomy (also known as Lane's Operation), total procto-colectomy and subtotal colectomy.

By "predicting medically refractory acute severe colitis", it is meant determining the likelihood that subject is will undergo medically refractory acute severe colitis following, the date at which the sample was obtained. By "a high risk of having medically refractory acute severe colitis" it is meant that the subject has a probability of at least 85% (i.e. 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100%) of having medically refractory acute severe colitis. A high risk of having medically refractory acute severe colitis in a subject refers to a subject who will not show a clinically significant relief in the disease when treated with an immunosuppressive drug treatment and therefore requiring colectomy. By "a low risk of having medically refractory acute severe colitis" it is meant that the subject has a probability of at least 85% (i.e. 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) of not having medically refractory acute severe colitis. A low risk of having medically refractory acute severe colitis in a subject refers to a subject who will show a clinically significant relief in the disease when treated with an immunosuppressive drug treatment.

Accordingly, the method of the present invention is particularly suitable for diagnosing susceptibility to medically refractory acute severe colitis. The method of the present invention is particularly suitable for avoiding colectomy or for determining whether the subject is elective for colectomy. As used herein, the phrase "elective for colectomy" refers to a subject who may choose to undergo the procedure of non-emergency colectomy based on physician and surgeon assessment following the performance of the method of the present invention. This differs from emergency colectomy, which is an acute intervention for subjects with acute illnesses or injuries and who require immediate medical attention. Finally, the method of present invention is particularly suitable for determining whether a subject suffering from acute severe colitis is eligible for a treatment with an immunosuppressive drug, in particular for determining whether the subject is eligible for a treatment with a corticosteroid.

Typically, the method of the present invention is performed once the subject is diagnosed with acute severe colitis.

As used herein, the term "sample" means any sample derived from the colon of the subject, which comprise mucosal cells. Said sample is obtained for the purpose of the in vitro evaluation. In a particular embodiment the sample results from an endoscopical biopsy performed in the colon of the subject. Said endoscopical biopsy may be taken from various areas of the colon. In another particular embodiment, the sample may be isolated from inflamed mucosa of the subject's colon.

Conventional methods and reagents for isolating R A from a sample comprise High Pure miR A Isolation Kit (Roche), Trizol (Invitrogen), Guanidinium thiocyanate-phenol- chloroform extraction, PureLink™ miRNA isolation kit (Invitrogen), PureLink Micro-to- Midi Total RNA Purification System (invitrogen), RNeasy kit (Qiagen), miRNeasy kit (Qiagen), Oligotex kit (Qiagen), phenol extraction, phenol-chloroform extraction, TC A/acetone precipitation, ethanol precipitation, Column purification, Silica gel membrane purification, Pure Yield™ RNA Midiprep (Promega), PolyATtract System 1000 (Promega), Maxwell® 16 System (Promega), SV Total RNA Isolation (Promega), geneMAG-RNA / DNA kit (Chemicell), TRI Reagent® (Ambion), RNAqueous Kit (Ambion), ToTALLY RNA™ Kit (Ambion), Poly(A)Purist™ Kit (Ambion) and any other methods, commercially available or not, known to the skilled person.

The term "miRNAs" refers to mature microRNA (non-coding small RNAs) molecules that are generally 21 to 22 nucleotides in length, even though lengths of 19 and up to 23 nucleotides have been reported. miRNAs are each processed from longer precursor RNA molecules ("precursor miRNA": pri-miRNA and pre-miRNA). Pri-miRNAs are transcribed either from non-protein-encoding genes or embedded into protein-coding genes (within introns or non-coding exons). The "precursor miRNAs" fold into hairpin structures containing imperfectly base-paired stems and are processed in two steps, catalyzed in animals by two Ribonuclease Ill-type endonucleases called Drosha and Dicer. The processed miRNAs (also referred to as "mature miRNA") are assembled into large ribonucleoprotein complexes (RISCs) that can associate them with their target mRNA in order to repress translation. All the miRNAs pertaining to the invention are known per se and sequences of them are publicly available from the data base http://www.mirbase.org/cgi-bin/mirna_summary.pl?org=hsa.

The expression level of one or more miRNA in the sample may be determined by any suitable method. Any reliable method for measuring the level or amount of miRNA in a sample may be used. Generally, miRNA can be detected and quantified from a sample (including fractions thereof), such as samples of isolated RNA by various methods known for mRNA, including, for example, amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g. , hybridization arrays (e.g. , microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, in situ hybridization, etc.), and sequencing- based methods (e.g. , next- generation sequencing methods, for example, using the Illumina or lonTorrent platforms). Other exemplary techniques include ribonuclease protection assay (RPA) and mass spectroscopy.

In some embodiments, RNA is converted to DNA (cDNA) prior to analysis. cDNA can be generated by reverse transcription of isolated miRNA using conventional techniques. miRNA reverse transcription kits are known and commercially available. Examples of suitable kits include, but are not limited to the mirVana TaqMan® miRNA transcription kit (Ambion, Austin, TX), and the TaqMan® miRNA transcription kit (Applied Biosystems, Foster City, CA). Universal primers, or specific primers, including miRNA- specific stem- loop primers, are known and commercially available, for example, from Applied Biosystems. In some embodiments, miRNA is amplified prior to measurement. In some embodiments, the expression level of miRNA is measured during the amplification process. In some embodiments, the expression level of miRNA is not amplified prior to measurement. Some exemplary methods suitable for determining the expression level of miRNA in a sample are described in greater hereinafter. These methods are provided by way of illustration only, and it will be apparent to a skilled person that other suitable methods may likewise be used.

Many amplification-based methods exist for detecting the expression level of miRNA nucleic acid sequences, including, but not limited to, PCR, RT-PCR, qPCR, and rolling circle amplification. Other amplification-based techniques include, for example, ligase chain reaction, multiplex ligatable probe amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification, RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art. A typical PCR reaction includes multiple steps, or cycles, that selectively amplify target nucleic acid species: a denaturing step, in which a target nucleic acid is denatured; an annealing step, in which a set of PCR primers (i.e., forward and reverse primers) anneal to complementary DNA strands, and an elongation step, in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target sequence. Typical PCR reactions include 20 or more cycles of denaturation, annealing, and elongation. In many cases, the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps. A reverse transcription reaction (which produces a cDNA sequence having complementarity to a miRNA) may be performed prior to PCR amplification. Reverse transcription reactions include the use of, e.g., a RNA -based DNA polymerase (reverse transcriptase) and a primer. Kits for quantitative real time PCR of miRNA are known, and are commercially available. Examples of suitable kits include, but are not limited to, the TaqMan® miRNA Assay (Applied Biosystems) and the mirVana™ qRT-PCR miRNA detection kit (Ambion). The miRNA can be ligated to a single stranded oligonucleotide containing universal primer sequences, a polyadenylated sequence, or adaptor sequence prior to reverse transcriptase and amplified using a primer complementary to the universal primer sequence, poly(T) primer, or primer comprising a sequence that is complementary to the adaptor sequence. In some embodiments, custom qRT-PCR assays can be developed for determination of miRNA levels. Custom qRT-PCR assays to measure miRNAs in a sample can be developed using, for example, methods that involve an extended reverse transcription primer and locked nucleic acid modified PCR. Custom miRNA assays can be tested by running the assay on a dilution series of chemically synthesized miRNA corresponding to the target sequence. This permits determination of the limit of detection and linear range of quantitation of each assay. Furthermore, when used as a standard curve, these data permit an estimate of the absolute abundance of miRNAs measured in the samples. Amplification curves may optionally be checked to verify that Ct values are assessed in the linear range of each amplification plot. Typically, the linear range spans several orders of magnitude. For each candidate miRNA assayed, a chemically synthesized version of the miRNA can be obtained and analyzed in a dilution series to determine the limit of sensitivity of the assay, and the linear range of quantitation. Relative expression levels may be determined, for example, according to the 2(- ΔΔ C(T)) Method, as described by Livak et ah, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔ C(T)) Method. Methods (2001) Dec;25(4):402-8.

In some embodiments, two or more miRNAs are amplified in a single reaction volume. For example, multiplex q-PCR, such as qRT-PCR, enables simultaneous amplification and quantification of at least two miRNAs of interest in one reaction volume by using more than one pair of primers and/or more than one probe. The primer pairs comprise at least one amplification primer that specifically binds each miRNA, and the probes are labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of multiple miRNAs.

Rolling circle amplification is a DNA-polymerase driven reaction that can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions (see, for example, Lizardi et al, Nat. Gen. (1998) 19(3):225-232; Gusev et al, Am. J. Pathol. (2001) 159(l):63-69; Nallur et al, Nucleic Acids Res. (2001) 29(23):E118). In the presence of two primers, one hybridizing to the (+) strand of DNA, and the other hybridizing to the (-) strand, a complex pattern of strand displacement results in the generation of over 10⁹ copies of each DNA molecule in 90 minutes or less. Tandemly linked copies of a closed circle DNA molecule may be formed by using a single primer. The process can also be performed using a matrix- associated DNA. The template used for rolling circle amplification may be reverse transcribed. This method can be used as a highly sensitive indicator of miRNA sequence and expression level at very low miRNA concentrations (see, for example, Cheng et al, Angew Chem. Int. Ed. Engl. (2009) 48(18):3268-72; Neubacher et al, Chembiochem. (2009) 10(8): 1289-91). miRNA may be detected using hybridization-based methods, including but not limited to hybridization arrays (e.g., microarrays), NanoString analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, and in situ hybridization. Microarrays can be used to measure the expression levels of large numbers of miRNAs simultaneously. Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre- made masks, photolithography using dynamic micromirror devices, inkjet printing, or electrochemistry on microelectrode arrays. Also useful are microfluidic TaqMan Low-Density Arrays, which are based on an array of microfluidic qRT-PCR reactions, as well as related microfluidic qRT-PCR based methods. In one example of microarray detection, various oligonucleotides (e.g., 200+ 5'- amino- modified-C6 oligos) corresponding to human sense miRNA sequences are spotted on three- dimensional CodeLink slides (GE Health/ Amersham Biosciences) at a final concentration of about 20 μMand processed according to manufacturer's recommendations. First strand cDNA synthesized from 20 μg TRIzol-purified total RNA is labeled with biotinylated ddUTP using the Enzo BioArray end labeling kit (Enzo Life Sciences Inc.). Hybridization, staining, and washing can be performed according to a modified Affymetrix Antisense genome array protocol. Axon B-4000 scanner and Gene-Pix Pro 4.0 software or other suitable software can be used to scan images. Non-positive spots after background subtraction, and outliers detected by the ESD procedure, are removed. The resulting signal intensity values are normalized to per-chip median values and then used to obtain geometric means and standard errors for each miRNA. Each miRNA signal can be transformed to log base 2, and a one-sample t test can be conducted. Independent hybridizations for each sample can be performed on chips with each miRNA spotted multiple times to increase the robustness of the data.

Microarrays can be used for the expression profiling of miRNAs. For example, RNA can be extracted from the sample and, optionally, the miRNAs are size- selected from total RNA. Oligonucleotide linkers can be attached to the 5' and 3' ends of the miRNAs and the resulting ligation products are used as templates for an RT-PCR reaction. The sense strand PCR primer can have a fiuorophore attached to its 5' end, thereby labeling the sense strand of the PCR product. The PCR product is denatured and then hybridized to the microarray. A PCR product, referred to as the target nucleic acid that is complementary to the corresponding miRNA capture probe sequence on the array will hybridize, via base pairing, to the spot at which the, capture probes are affixed. The spot will then fluoresce when excited using a microarray laser scanner. The fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA. Total RNA containing the miRNA extracted from the sample can also be used directly without size-selection of the miRNAs. For example, the RNA can be 3' end labeled using T4 RNA ligase and a fiuorophore-labeled short RNA linker. Fiuorophore- labeled miRNAs complementary to the corresponding miRNA capture probe sequences on the array hybridize, via base pairing, to the spot at which the capture probes are affixed. The fluorescence intensity of each spot is then evaluated in terms of the number of copies of a particular miRNA, using a number of positive and negative controls and array data normalization methods, which will result in assessment of the level of expression of a particular miRNA. Several types of microarrays can be employed including, but not limited to, spotted oligonucleotide microarrays, pre-fabricated oligonucleotide microarrays or spotted long oligonucleotide arrays. miRNAs can also be detected without amplification using the nCounter Analysis System (NanoString Technologies, Seattle, WA). This technology employs two nucleic acid- based probes that hybridize in solution (e.g., a reporter probe and a capture probe). After hybridization, excess probes are removed, and probe/target complexes are analyzed in accordance with the manufacturer's protocol. nCounter miRNA assay kits are available from NanoString Technologies, which are capable of distinguishing between highly similar miRNAs with great specificity. The basis of the nCounter® Analysis system is the unique code assigned to each nucleic acid target to be assayed (International Patent Application Publication No. WO 08/124847, U.S. Patent No. 8,415,102 and Geiss et al. Nature Biotechnology. 2008. 26(3): 317-325; the contents of which are each incorporated herein by reference in their entireties). The code is composed of an ordered series of colored fluorescent spots which create a unique barcode for each target to be assayed. A pair of probes is designed for each DNA or RNA target, a biotinylated capture probe and a reporter probe carrying the fluorescent barcode. This system is also referred to, herein, as the nanoreporter code system. Specific reporter and capture probes are synthesized for each target. The reporter probe can comprise at a least a first label attachment region to which are attached one or more label monomers that emit light constituting a first signal; at least a second label attachment region, which is non-over- lapping with the first label attachment region, to which are attached one or more label monomers that emit light constituting a second signal; and a first target- specific sequence. Preferably, each sequence specific reporter probe comprises a target specific sequence capable of hybridizing to no more than one gene and optionally comprises at least three, or at least four label attachment regions, said attachment regions comprising one or more label monomers that emit light, constituting at least a third signal, or at least a fourth signal, respectively. The capture probe can comprise a second target-specific sequence; and a first affinity tag. In some embodiments, the capture probe can also comprise one or more label attachment regions. Preferably, the first target- specific sequence of the reporter probe and the second target- specific sequence of the capture probe hybridize to different regions of the same gene to be detected. Reporter and capture probes are all pooled into a single hybridization mixture, the "probe library". The relative abundance of each target is measured in a single multiplexed hybridization reaction. The method comprises contacting the tumor sample with a probe library, such that the presence of the target in the sample creates a probe pair - target complex. The complex is then purified. More specifically, the sample is combined with the probe library, and hybridization occurs in solution. After hybridization, the tripartite hybridized complexes (probe pairs and target) are purified in a two-step procedure using magnetic beads linked to oligonucleotides complementary to universal sequences present on the capture and reporter probes. This dual purification process allows the hybridization reaction to be driven to completion with a large excess of target-specific probes, as they are ultimately removed, and, thus, do not interfere with binding and imaging of the sample. All post hybridization steps are handled robotically on a custom liquid- handling robot (Prep Station, NanoString Technologies). Purified reactions are typically deposited by the Prep Station into individual flow cells of a sample cartridge, bound to a streptavidin-coated surface via the capture probe,electrophoresed to elongate the reporter probes, and immobilized. After processing, the sample cartridge is transferred to a fully automated imaging and data collection device (Digital Analyzer, NanoString Technologies). The expression level of a target is measured by imaging each sample and counting the number of times the code for that target is detected. For each sample, typically 600 fields-of-view (FOV) are imaged (1376 X 1024 pixels) representing approximately 10 mm2 of the binding surface. Typical imaging density is 100- 1200 counted reporters per field of view depending on the degree of multiplexing, the amount of sample input, and overall target abundance. Data is output in simple spreadsheet format listing the number of counts per target, per sample. This system can be used along with nanoreporters. Additional disclosure regarding nanoreporters can be found in International Publication No. WO 07/076129 and WO07/076132, and US Patent Publication No. 2010/0015607 and 2010/0261026, the contents of which are incorporated herein in their entireties. Further, the term nucleic acid probes and nanoreporters can include the rationally designed (e.g. synthetic sequences) described in International Publication No. WO 2010/019826 and US Patent Publication No.2010/0047924, incorporated herein by reference in its entirety.

Mass spectroscopy can be used to quantify miRNA using RNase mapping. Isolated RNAs can be enzymatically digested with RNA endonucleases (RNases) having high specificity (e.g., RNase Tl, which cleaves at the 3'-side of all unmodified guanosine residues) prior to their analysis by MS or tandem MS (MS/MS) approaches. The first approach developed utilized the on-line chromatographic separation of endonuclease digests by reversed phase HPLC coupled directly to ESTMS. The presence of posttranscriptional modifications can be revealed by mass shifts from those expected based upon the RNA sequence. Ions of anomalous mass/charge values can then be isolated for tandem MS sequencing to locate the sequence placement of the posttranscriptionally modified nucleoside. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used as an analytical approach for obtaining information about posttranscriptionally modified nucleosides. MALDI-based approaches can be differentiated from ESTbased approaches by the separation step. In MALDI-MS, the mass spectrometer is used to separate the miRNA. To analyze a limited quantity of intact miRNAs, a system of capillary LC coupled with nanoESI- MS can be employed, by using a linear ion trap-orbitrap hybrid mass spectrometer (LTQ Orbitrap XL, Thermo Fisher Scientific) or a tandem-quadrupole time- of-flight mass spectrometer (QSTAR® XL, Applied Biosystems) equipped with a custom-made nanospray ion source, a Nanovolume Valve (Valco Instruments), and a splitless nano HPLC system (DiNa, KYA Technologies). Analyte/TEAA is loaded onto a nano-LC trap column, desalted, and then concentrated. Intact miRNAs are eluted from the trap column and directly injected into a CI 8 capillary column, and chromatographed by RP-HPLC using a gradient of solvents of increasing polarity. The chromatographic eluent is sprayed from a sprayer tip attached to the capillary column, using an ionization voltage that allows ions to be scanned in the negative polarity mode.

Additional methods for miR A detection and measurement include, for example, strand invasion assay (Third Wave Technologies, Inc.), surface plasmon resonance (SPR), cDNA, MTDNA (metallic DNA; Advance Technologies, Saskatoon, SK), and single- molecule methods such as the one developed by US Genomics. Multiple miRNAs can be detected in a microarray format using a novel approach that combines a surface enzyme reaction with nanoparticle- amplified SPR imaging (SPRI). The surface reaction of poly(A) polymerase creates poly(A) tails on miRNAs hybridized onto locked nucleic acid (LNA) microarrays. DNA-modified nanoparticles are then adsorbed onto the poly(A) tails and detected with SPRI. This ultrasensitive nanoparticle-amplified SPRI methodology can be used for miRNA profiling at attamole levels. miRNAs can also be detected using branched DNA (bDNA) signal amplification (see, for example, Urdea, Nature Biotechnology (1994), 12:926- 928). miRNA assays based on bDNA signal amplification are commercially available. One such assay is the QuantiGene® 2.0 miRNA Assay (Affymetrix, Santa Clara, CA). Northern Blot and in situ hybridization may also be used to detect miRNAs. Suitable methods for performing Northern Blot and in situ hybridization are known in the art. Advanced sequencing methods can likewise be used as available. For example, miRNAs can be detected using Illumina ® Next Generation Sequencing (e.g. Sequencing-By-Synthesis or TruSeq methods, using, for example, the HiSeq, HiScan, GenomeAnalyzer, or MiSeq systems (Illumina, Inc., San Diego, CA)). miRNAs can also be detected using Ion Torrent Sequencing (Ion Torrent Systems, Inc., Gulliford, CT), or other suitable methods of semiconductor sequencing.

Typically, the predetermined reference value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the expression level of the selected miRNA in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the expression level of the selected miRNA in a group of reference, one can use algorithmic analysis for the statistic treatment of the expression levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPO WER. S AS , DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

A further object of the invention relates to a kit for performing the method of the present invention, wherein said kit comprises means for measuring the expression levels of the miRNAs of the invention in the sample obtained from the patient. The kits may include probes, primers macroarrays or microarrays as above described. For example, the kit may comprise a set of miRNA probes as above defined, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers. The kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards. Alternatively the kit of the invention may comprise amplification primers (e.g. stem- loop primers) that may be pre- labelled or may contain an affinity purification or attachment moiety. The kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.

In some embodiments, labels, dyes, or labeled probes and/or primers are used to detect amplified or unamp lifted miRNAs. The skilled artisan will recognize which detection methods are appropriate based on the sensitivity of the detection method and the abundance of the target. Depending on the sensitivity of the detection method and the abundance of the target, amplification may or may not be required prior to detection. One skilled in the art will recognize the detection methods where miRNA amplification is preferred. A probe or primer may include standard (A, T or U, G and C) bases, or modified bases. Modified bases include, but are not limited to, the AEGIS bases (from Eragen Biosciences), which have been described, e.g., in U.S. Pat. Nos. 5,432,272, 5,965,364, and 6,001,983. In certain aspects, bases are joined by a natural phosphodiester bond or a different chemical linkage. Different chemical linkages include, but are not limited to, a peptide bond or a Locked Nucleic Acid (LNA) linkage, which is described, e.g., in U.S. Pat. No. 7,060,809. In a further aspect, oligonucleotide probes or primers present in an amplification reaction are suitable for monitoring the amount of amplification product produced as a function of time. In certain aspects, probes having different single stranded versus double stranded character are used to detect the nucleic acid. Probes include, but are not limited to, the 5'-exonuclease assay {e.g., TaqMan™) probes (see U.S. Pat. No.5, 538, 848), stem-loop molecular beacons (see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517), stemless or linear beacons (see, e.g., WO 9921881, U.S. Pat. Nos. 6,485,901 and 6,649,349), peptide nucleic acid (PNA) Molecular Beacons (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g. U.S. Pat. No. 6,329,144), non- FRET probes (see, e.g., U.S. Pat. No. 6,150,097), Sunrise™/AmplifiuorB™ probes (see, e.g., U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion™ probes (see, e.g., U.S. Pat. No. 6,589,743), bulge loop probes (see, e.g., U.S. Pat. No. 6,590,091), pseudo knot probes (see, e.g., U.S. Pat. No. 6,548,250), cyclicons (see, e.g., U.S. Pat. No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (see, e.g. , U.S. Pat. No. 6,596,490), PNA light-up probes, antiprimer quench probes (Li et al, Clin. Chem.53:624-633 (2006)), self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901. In some embodiments, one or more of the primers in an amplification reaction can include a label. In yet further embodiments, different probes or primers comprise detectable labels that are distinguishable from one another. In some embodiments a nucleic acid, such as the probe or primer, may be labeled with two or more distinguishable labels. In some aspects, a label is attached to one or more probes and has one or more of the following properties: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g., FRET (Fluorescent Resonance Energy Transfer); (iii) stabilizes hybridization, e.g., duplex formation; and (iv) provides a member of a binding complex or affinity set, e.g. , affinity, antibody- antigen, ionic complexes, hapten-ligand (e.g. , biotin-avidin). In still other aspects, use of labels can be accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods. Labels include, but are not limited to: light-emitting, light- scattering, and light- absorbing compounds which generate or quench a detectable fluorescent, chemiluminescent, or bio luminescent signal (see, e.g. , Kricka, L., Nonisotopic DNA Probe Techniques, Academic Press, San Diego (1992) and Garman A., Non- Radioactive Labeling, Academic Press (1997).). A dual labeled fluorescent probe that includes a reporter fluorophore and a quencher fluorophore is used in some embodiments. It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished. In some embodiments, labels are hybridization- stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g. , intercalators and intercalating dyes (including, but not limited to, ethidium bromide and SYBR-Green), minor-groove binders, and cross-linking functional groups (see, e.g. , Blackburn et al., eds. "DNA and RNA Structure" in Nucleic Acids in Chemistry and Biology (1996)).

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1. Supervised hierarchical clustering of 30 samples based on their response to treatment. The dendrogram was obtained based on the expression data obtained by microarray of 51 pre-microRNAs and microRNAs that differ significantly with a fold change of at least 1.3 between the 2 subgroups of responders. There is a cluster of 12/12 steroid-responsive (P -value <0.00001). CS; cortico-sensitive, CA treatment with ciclosporin, IF; treatment with Infliximab Figure 2. Hierarchical clustering of the 30 samples supervised on the 9 pre- microARN and microARN, on their response to the treatment. CS; cortico-sensitive, CA treatment with ciclosporin, IF; treatment with Infliximab

EXAMPLE:

Material & Methods

Patients and judgment criteria

The study population was recruited from two centres: Gastroenterology, IBD Beaujon

Hospital (AP-HP, Clichy) and gastroenterology department of the hospital of Haut-Leveque (CHU de Bordeaux, Pessac). Inclusion criteria were: age over 18 years, ulcerative colitis proved as clinical and para-clinical practice, Lichtiger score greater than 10 at the time of hospitalization, double-sigmoidoscopy with colonic biopsies within the 5 days following admission. Only biopsies with histological grading of 3 or 4 were analyzed. Patient selection was made retrospectively from databases pathology services of the 2 centers and with the clinical follow-up patient records. 12 of the 30 patients analyzed, were part of the study led by CYSIF GETAID and had data collected prospectively. All patients were analyzed uniformly according to the criteria of the CYSIF study. Patients had received 1 to 3 lines of treatment. Intravenous corticosteroids routinely constituted the first line of treatment. The outcomes were similar to CYSIF study. Treatment failure was defined by the following criteria: absence of clinical response at day 7 (Lichtiger score> 10), relapse before 3 months (re-ascent of Lichtiger score more than 3 points for at least 3 consecutive days), need for a new line of treatment, discontinuation of therapy due to a major side effect, colectomy or death within 3 months. Clinical response was defined as a Lichtige score of less than 10 at day 5 for at least 2 consecutive days, with a decrease of at least 3 points from the initial score.

RNA extraction from paraffin-embedded tissue Paraffin embedded biopsies were recovered in pathology services. The RNA extraction was performed using the kit RecoverAll™ Total Nucleic Acid of Ambion®. The initial sample consisted of 7 chips 10 microns thick and dewaxed. In accordance with the kit protocol, they were subjected to a treatment with protease and a DNase. The quantity and purity of RNA extracted for each sample were evaluated by spectrophotometry (Nanodrop™ Spectrophotometer) .

Measuring the expression of microRNA by RT-qPCR

RT-qPCR was performed with TaqMan probes specific to human miRNAs. CDNA was synthesized from 10 ng RNA using primers specific microRNAs. RT-qPCR was performed following the manufacturer's instructions, from total RNA aliquots containing the equivalent of 1.3 ng of cDNA and 480 through the LightCycler® (Roche Diagnostics).

Measuring the expression of microRNAs by microarray

The level of expression of microRNAs was measured using a chip microRNAs. This is the GeneChip miRNA Arrays Affymetrix® chip analyzing the level of expression of more than 30000 more than 3000 miRNAs pre-microRNAs and human microRNA. These analyzes were carried out under the responsibility of Sebastien Jacques (MSc) in genomics platform Cochin Institute (rue du Faubourg Saint- Jacques, 75014 Paris, France).

Statistical analyses

Statistical analyzes were performed with GraphPad Prism software and dChip. Comparison of clinical characteristics of patients was carried out by Chi2 tests, Fischer exact test and by Student's t test, respectively, for the qualitative and quantitative variables. The hierarchical clustering were obtained using the dChip 2010 software (http://www.hsph.harvard.edu/cli/complab/dchip/). The difference in expression between subgroups were analyzed using the t test dChip.

Results

Patients were retrospectively selected after analysis of databases of the centres were the study was performed, and after analysis of medical records. A total of 41 patients met the criteria for clinical inclusion and 30 patients were analyzed. The characteristics of the patients are shown in Table 1. Charateristics N=30

Male 13 / 30 (43.33%)

Mean age at diagnostic (years) 31.33 (σ = 4.14)

Corticoids subsequently to the biopsy 21 / 30 (70 ,00%)

IBD family history 7 /30 (23.33%)

Appendectomy 1 / 28 (3.57%)

E3 (Inflammation spreading beyond left angle) 23 / 28 (82.14%)

Extra digestive symptoms 3 / 30 (10.00%)

Mean Lichtiger score at admission 12.83 (σ = 2.38)

Deep ulcerations 8 / 30 (26.67%)

Smoking never / weaned / active 19 / 5 / 5

Table 1: characteristics of the patients Starting from the clinical samples and following 6 criteria (response to corticosteroids, corticosteroids subsequently to biopsy, smoking, presence of deep ulcerations, patients with multiple CAG and family history of IBD) we built from the raw expression results of 3372 pre-microRNAs a hierarchical cluster of samples by the Pearson method on a single clinical endpoint which was the response to corticosteroids. This leads to a cluster of samples significantly enriched in steroid-responsive patients (7 of 8 patients) (for 13 steroid- responsive patients out of 30 in total) (P -value = 0.014250).

In a second step, we compared the expression of microRNAs in the 2 subgroups of patients (responders and non responders to corticosteroids) to keep only the pre-microRNAs and microRNA that differed with a fold change of at least 1.3 and significantly differentially expressed (t-test, P-value <0.05). We found 51 miRNAs and pre-miRNAs that were differentially expressed. By making a new hierarchical clustering, the expression of these 51 genes, two clusters were defined and corresponded almost perfectly to the 2 subgroups of responders to corticosteroids. So we got a cluster of 12 samples, all corresponding to steroid- responsive patients to 13 of 30 total (P-value <0.00001) (Figure 1). When we reduced the number of pre-microRNAs and microRNA to 9 (those that differ between the two subgroups with a P-value <0.001) we obtained a cluster of 14 samples significantly enriched in steroid- responsive patients (12 patients 14) (P-value = 0.0014) (Figure 2).

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method for predicting medically refractory acute severe colitis in a subject comprising the steps consisting of i) determining in a sample obtained from the subject the expression levels of miR-1825, miR-938, miR-4699, miR-23c, miR-4659a- 3p, miR-4477a, miR-320b-l, miR-3128, and miR-4755, comparing the expression levels determined at step i) with their corresponding predetermined reference values and ii) concluding that the subject has a high risk of having medically refractory acute severe colitis when the expression level of miR-1825 and miR-938 are lower than their respective predetermined reference values and when the expression levels of miR-4699, miR-23c, miR-4659a-3p, miR-4477a, miR-320b-l, miR-3128, and miR-4755 are higher than their respective predetermined reference values, or concluding that the subject has a low risk of having medically refractory acute severe colitis when the expression level of miR-1825 and miR-938 are higher than their respective predetermined reference values and when the expression levels of miR-4699, miR-23c, miR-4659a-3p, miR-4477a, miR-320b-l, miR-3128, and miR-4755 are lower than their respective predetermined reference values.

2. A method of treating acute severe colitis in a subject in need thereof comprising the steps of i) determining whether has a high or a low risk of having medically refractory acute severe colitis by the method of claim 1 and ii) administering the subject with an immunosuppressive drug when a low risk of having medically refractory acute severe colitis is determined at step i) or subjecting the subject to colectomy if a high risk of having medically refractory acute severe colitis is determined at step i).

3. The method of claim 2 wherein the immunosuppressive drug is a corticosteroid. 4. The method of claim 3 wherein the corticosteroid is selected from the group consisiting of cortisone, Cortisol, hydrocortisone (1 ip,17-dihydroxy, 21- (phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxycortisone, dexamethasone (21 -(acetyloxy)-9-fluoro- 1 β, 17-dihydroxy- 16a-m-ethylpregna- 1 ,4- diene-3,20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-l 1-β, 17,21, trihydroxy-16P- methylpregna-1,

4 diene-3,20-dione 17,21-dipropionate). Other examples of corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort and betamethasone, corticosteroids, for example, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.

5. The method of claim 2 wherein the immunosuppressive drug in an anti-TNFalpha drug.

6. The method of claim 5 wherein the anti-TNFalpha drug is selected from the group consisting of infliximab, human anti-TNF monoclonal antibody adalimumab, etanercept, certolizumab pegol, golimumab, CDP 571, and CDP 870.

7. The method of claim 2 wherein the immunosuppressive drug is ciclosporin.